Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

Abstract

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

Figure 1. Disruption of an eGFP transgene in G. bimaculatus using designed ZFNs.

(a) Illustration of the binding and function of ZFNs and TALENs. Following the introduction of double-stranded breaks (DSB) in the DNA between targeted binding sites, these breaks are either repaired and digested again, or NHEJ can occur and deletions or insertions can result. In the latter case, gene function of the targeted gene is disrupted, and subsequent binding of ZFNs or TALENs does not result in FokI dimerization or the generation of DSBs. (b) Structure of the pBGact-eGFP transgene that is under the transcriptional control of the Gryllus cytoplasmic actin promoter (Gact) and flanked by 5′ and 3′ inverted terminal repeats (ITRs). The 9-bp binding sites for the ZFNs, eGFP-R and eGFP-L are in the boxes shown. (c) Detection of induced mutations into genomic DNA cleaved by Surveyor nuclease that was collected from embryos injected with both eGFP-R and eGFP-L mRNAs or eGFP-R mRNA alone (control) at 7 days postinjection. A product cleaved by Surveyor nuclease was detected in the genomic DNA from embryos injected with eGFP-R/L mRNAs (arrowhead). (d) Bright field (left panel) and fluorescence (right panel) phenotypes of G₁ crickets injected with eGFP ZFN mRNA at 2 days after egg laying. While the embryos appeared to develop normally, some embryos did not exhibit eGFP fluorescence (indicated with arrows). Scale bar, 2.5 mm. (e) Sequence analysis of eGFP mutant alleles induced by eGFP ZFN mRNAs. Wild-type (WT) sequences are shown above the mutant sequences containing deletions (indicated with dashes) and/or insertions (shown in red letters). Asterisks indicate knockout mutations that included frame-shift and stop codon insertions (underlined).

Figure 2. Design and application of ZFNs and TALENs targeted to Gb′lac2.

(a) Structure of the Gb′lac2 gene, with open boxes representing exons mapped on the Gryllus genomic sequence. Black bars indicate Cu-oxidase domains 1–3. The grey region of the fifth exon is expanded to provide the gene sequence that includes the 9 or 17 bp ZFN and TALEN binding sites, respectively (shown in boxes). (b) Dose-dependent toxicity of Gb′lac2 ZFN mRNAs in cricket embryos. The percentages of dead (black), deformed (grey) and normal (white) embryos at 2 days postinjection are shown. The control was embryos injected with Lac2-L ZFN mRNA alone. The percentage of embryos that developed normally increased with decreasing concentrations of Gb′lac2 ZFN mRNAs (0/43 for 1 μg μl⁻¹ each, 6/51 for 10 ng μl⁻¹ each, 19/50 for 1 ng μl⁻¹ each, 13/25 for 100 pg μl⁻¹ each and 11/21 for control). (c) Using a Surveyor nuclease assay, mutations in Gb′lac2 were detected following the microinjection of crickets with Gb′lac2 ZFN mRNAs. Products cleaved by Surveyor nuclease (indicated with arrowheads) were detected at 1 μg μl⁻¹, 100 ng μl⁻¹, 10 ng μl⁻¹ and 1 ng μl⁻¹ of Lac2 ZFN injected. (d) Imaging of control and Gb′lac2 ZFN final-instar nymphs. Mutagenesis of somatic cells in G₀ crickets was detected based on the presence of a white spot phenotype by epidermal cells (indicated with arrows).

Figure 3. Isolation of homozygous mutants after injection of Gb′lac2 ZFN mRNAs into crickets.

(a) An illustration of the scheme used to isolate homozygous mutations in an endogenous gene. Mutagenized G₀ adults were crossed to wild-type (WT) adults, and G₁ embryos were checked for heterozygous mutations in a first round of screening using the Surveyor nuclease. Positive G₁ individuals were developed into final-instar nymphs and subjected to a second round of screening with Surveyor nuclease assays. Positive G₁ adults were then crossed for each strain to obtain homozygous mutants in G₂ generation. (b) Germline mutations in G₀ crickets were detected in a first-round screening of G₁ embryos using Surveyor nuclease assays. The arrowhead indicates the product cleaved by the Surveyor nuclease in the f-2 line. Arrow indicates 200-bp size marker. (c,d) Isolation of heterozygous mutants from G₁ crickets in f-2 (c) and f-17 (d) lines detected in the second round of screening. Heterozygous mutant G₁ crickets were detected in a second round of screening of T3 legs from G₁ final-instar nymphs. Nymphs that harboured mutations caused by ZFNs are underlined. Arrowheads indicate the products generated from Surveyor nuclease assays. Asterisks indicate the presence of homoduplex PCR products from mutant alleles. Arrows indicate 200-bp size marker. (e) Sequence analysis of Gb′lac2 mutant alleles induced by ZFNs. Wild-type sequences are shown above the mutant sequences containing deletions (indicated with dashes) and/or insertions (shown in red letters). Boxed sequences represent ZFN-binding sites. Asterisks indicate frame-shift mutations are present. (f–h) Phenotypes of G₂ heterozygous and homozygous mutant crickets 1 day after hatching. Control first-instar nymphs were coloured black until 1 day posthatching, while heterozygous mutants were still grey in colour and homozygous mutants were white. (i) Genotyping of G₂ heterozygous and homozygous mutants. In Surveyor nuclease assays, cleavage products were detected in heterozygous mutants (indicated with an arrowhead), but not in WT or homozygous mutants (right panel). The asterisk indicates heteroduplex PCR products from WT and mutant alleles were present. Arrow indicates 200-bp size marker.

Figure 4. Isolation of homozygous mutants induced by the microinjection of Gb′lac2 TALEN mRNA into crickets.

(a) Toxicity of Gb′lac2 TALEN mRNAs in cricket embryos was detected. Percentages of dead (black), deformed (grey) and normal (white) embryos are shown at 2 days postinjection. The control included embryos injected with Lac2-L TALEN mRNA alone. The percentage of embryos that developed normally after microinjection of Gb′lac2 TALEN mRNAs (68%, 82/121) was comparable to control (64%, 30/47). (b) Germline mutations in G₀ crickets were detected in a first-round screening of G₁ embryos. Arrowheads indicate the cleavage products generated in Surveyor nuclease assays of the f-4 line. Arrow indicates 200-bp size marker. (c,d) A second round of Surveyor nuclease assays was performed to identify heterozygous mutants present in G₁ crickets from the f-4 (c) and f-11 (d) lines. Adults that harboured mutations caused by TALENs are underlined, while arrowheads indicate cleavage products from Surveyor nuclease assays. Arrows indicate 200-bp size marker. (e–g) Phenotypes of G₂ heterozygous and homozygous Gb′lac2 mutants generated with TALENs 1 day after hatching. Control first-instar nymphs were coloured black, while heterozygous mutants were still grey in colour and homozygous mutants were white. (h) Sequence analysis of Gb′lac2 mutant alleles induced by TALENs. The wild-type (WT) sequence is shown above the mutant sequences containing deletions (indicated with dashes) and/or insertions (shown in red letters). Boxed sequences represent TALEN-binding sites.